Small, truncated chromosomes have been associated with certain diseases and occasionally identified in the normal populations, as well as generated experimentally. These mitotically stable mini-chromosomes are very valuable for the identification and characterization of functional chromosome elements and could be converted to manipulable human artificial chromosomes (HAC). We acquired four cell lines containing mini-chromosomes, three of them from normal individuals and one generated experimentally. The mini-chromosomes range from 3 to 10 mb in size and their chromosomal origins were determined. Microdissection of the mini-chromosomes was performed, and the microdissected DNA was used to screen chromosome-specific cosmid libraries. These studies have revealed that these mini-chromosomes contain limited euchromatic sequences and truncated centromeres. Further analysis at the sequence level is in progress which may identify functional subfamilies of the repeated satellite DNA at human centromeres. Additional physical mapping analysis will help to define other necessary sequences for chromosome functions. Selectable markers will be inserted into the mini-chromosomes through homologous recombination and the feasibility of using these mini-chromosomes as HAC vectors will be tested.